Real-time nanoscale investigation of spore coat assembly in Bacillus subtilis.

Spores of Bacillaceae, ubiquitous in soil, can withstand extreme conditions virtually indefinitely. The spore coat, one of the most sophisticated multiprotein complexes built by bacteria, protects the spore from environmental stresses and predators. In Bacillus subtilis, assembly of the multilayered coat initiates at the forespore pole and is perceived as a stable process that progresses continuously until covering the entire forespore surface. In contrast, in Bacillus cereus, coat formation initiates in the midspore region and extends outward to the spore poles. Using Structured Illumination Microscopy, we monitored B. subtilis coat development in real-time at the single-sporangium level with lateral resolution of 70 nm. We found that late-synthesized proteins from the innermost coat layers first assemble in the midspore region and are subsequently displaced toward the poles. This process is coupled with a unique redistribution of pre-assembled coat material across the forespore surface and influenced by outer coat development, highlighting a dynamic interplay in coat layer co-construction.